Illumina HiSeq 2000 sequencing; GSM2432382: F2 Sample 1; Arabidopsis thaliana; RNA-Seq

F2 Sample 1

In this study, we describe the impact of genetic variation on transcript abundance in an F2 population of Arabidopsis thaliana. The RNA-seq resource generated by this study is suitable for expression quantitative trait locus (eQTL) mapping. From the aligned RNA-seq reads, and available genomic data for each of the parents of the cross, we imputed the genomes of each F2 individual (to allow genetic mapping of RNA abundance traits; briefly, genetic differences in aligned RNA-seq reads were used to impute each F2 genome). Our results show that heritable differences on gene expression can be detected using F2 populations (that is, single F2 plants), and shed light on the control of expression differences among strains of this reference plant. Overall design: 183 samples consisting of single F2 plants of a cross between Arabidopsis thaliana accessions 8230 and 6195 were generated. For each sample, RNA was collected from the aerial shoot at the 9th true leaf stage, and Illumina mRNA-seq libraries were constructed. Using these libraries, 50 bp single end RNA-seq Illumina reads were generated for each sample, and used to quantify gene expresison in each individual. The resulting expression phenotypes are suitable for genetic mapping of the control of gene expression differences in the species.

Genetic influences on gene expression in Arabidopsis thaliana

Submitted by Gene Expression Omnibus on 16-MAR-2017

Briefly, individual frozen plants were ground in liquid nitrogen using a mortar and pestle and RNA was isolated using PureLink Plant RNA Reagent (#12322-012, Invitrogen, Carlsbad, CA, USA) following the manufacturers instructions. Precipitated RNA was resuspended in RNAsecure Resuspension Solution (#AM7010, Ambion; Life Technologies, Carlsbad, CA, USA) following manufacturers guidelines to prevent RNA degradation. Contaminating genomic DNA was removed by incubating samples with Turbo DNase (#AM2238, Ambion) for 15 min at 37°C. Finally, RNA was precipitated using 2.5 vol 100% ethanol, 0.1 volume 5M Ammonium Acetate and 1µl glycogen (5 mg/ml) and resuspended in RNase-free water. RNA-seq libraries were constructed using the Illumina TruSeq RNA Sample Preparation Kit V2 (# 15026495 Rev. C). Briefly, poly-A mRNA was purified from 1 µg of isolated total RNA using poly-T oligo-attached magnetic beads. Purified RNA was fragmented into 120-210 bp fragments (average size ~155 bp) using divalent cations at 94°C for 8 minutes. After fragmentation, first strand cDNA synthesis was performed using random primers and reverse transcriptase, followed by second strand cDNA synthesis using DNA polymerase I and RNase H. The cDNA fragments then went through end repair and overhang A addition reactions followed by the ligation of the RNA Adapters. Finally, the products were purified and enriched using 12 PCR cycles to create the final cDNA sequencing library.

Genetic influences on gene expression in Arabidopsis thaliana

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